Top 10 Crispiest CRISPR Applications*

There is a heady and hysterical goldrush to CRISPR ALL THE THINGS. And with good reason. These are not your grandpa’s GMOs.

“Second-generation” genome-editing tools can now precisely convert a single base into another without the need for double strand break or incorporating a gene from another organism. At the drop of a “nickase,” C can be converted to T, and A to G, generating a STOP codon and abolishing the need for complex knockout — strategies. (Review CRISPR fundamentals here.)

Like the immeasurable heaven of the Laniakea supercluster, the applications of CRISPR seem to know no bounds. But, the most exciting applications for CRISPR have little to do with gene editing. At the rate of CRISPR publications (1000s per year), you may forgive yourself for not being able to stay up on the literature.

I have compiled some of my favorite (for about a minute) CRISPR applications. The breathless future of CRISPR means these will likely be overturned faster than an ubiquitinated protein.

1. Gene “Scaring” for Massively Parallel Whole Organism or Single Cell Lineage Tracing- In the absence of a template for homologous repair, Cas9 produces short insertions or deletions (indels) at its target sites, which are variable in their length and position. This generates hundreds of scars- essentially “barcoding” each cell (either in a transgenic reporter line or whole organism) in order to simultaneously trace the lineage of and profile the transcriptome of thousands of single cells. Massively parallel profiling strategies are being adopted across the board for large-scale functional screenings.

3. RNA Editing for Programmable A to I Replacement- New ‘REPAIR’ system edits RNA, rather than DNA; and has potential to treat diseases without permanently affecting the genome. REPAIR has the ability to target individual RNA letters, or nucleosides, switching adenosines to inosines (read as guanosines by the cell). These letters are involved in single-base changes known to regularly cause disease in humans. In human disease, a mutation from G to A is extremely common; these alterations have been implicated in, for example, cases of focal epilepsy, Duchenne muscular dystrophy, and Parkinson’s disease.

4. RNA Scissors to Regulate Translation- The discovery of another component of the CRISPR system, a special pair of RNA scissors- allows for us to now regulate protein translation too. Just announced in February of 2018- I am speculating that this will quickly become a multi-functional tool.

5. Light-Inducible ON/Off Toggle Switch — Optogenetics uses genetically encoded tools, such as microbial opsins, to control cellular activities using light. In 2015, scientists combined CRISPR and optogenetics techniques to develop a variety of photoactivatable CRISPR tools. These tools allow scientists to use light to externally control the location, timing, and reversibility of the genome editing process.

6. CRISPR interference (CRISPRi) and CRISPR Activation (CRISPRa)- In CRISPRi, a steric block halts transcript elongation by RNA polymerase, resulting in the knockdown or repression of the target gene. CRISPRa technology allows for overexpression of genes through recruitment of transcription activators for targeted gene activation in their endogenous context and is applicable to both coding and noncoding genes.

7. Changing the Epigenetic Signature — Epigentics is itself a new science. There are multiple layers of epigenetic regulatory mechanisms operating in the genome. Among the well-described ones are DNA methylation, histone posttranslational modifications, and non-coding RNAs (short and long). Guiding Cas9 to a target sequence with a –methylase or –acetylase can increase or reduce DNA and histone methylation, and acetylation on lysine tails.

8. Live–Cell Chromatin Imaging- The organization of chromatin in 3D space plays a critical role in regulating gene expression. Multi-colored tracking of native (ie. unfixed live cell) chromatin loci has started to illuminate the positioning of transcriptionally active and inactive regions of chromatin in the 3D nuclear space throughout the cell cycle.

9. Synthetic Chromatin Biology: Do you remember the scene in the movie Contact when S.R. Hadden describes how the registration marks line the images up in three-dimensional space reveal the primer? That is like a chromatin loop. Chromatin loop-structures bring distant pieces of DNA (enhancers and promoters) close to regulate gene expression. These loops can be directly engineered with CRISPR to either promote or inhibit the enhancer — promoter activity. Synthetic DNA regulation systems of unprecedented sophistication can be created with by combining spatiotemporal layers of chromatin regulation.

10. Gene Drives- Simply put, a gene drive gives a phenotype an advantage, a “drive” that allows the gene to be passed on to more than 50% of its offspring, as in conventional reproduction. In several examples with mosquitos researchers were able to push a gene that inhibits the transmission of malaria to more than 95% of the progeny.

These are my current top ten but, new and intriguing developments are already on the horizon. For example, the “docking” sites for CRISPR are genome segments that have at one end a specific three-base trio: N, where N is any of DNA’s four bases, followed by two guanines (Gs). Only about one-sixteenth of the 3.2-billion-base human genome has the right sequence. In February of 2018, a newly engineered xCas9 promises to release the PAM restrictions and allow access to 4X more genomic docking sites with greater precision and less off target effects.